A receptor for t-PA was previously described on human umbilical vein endothelial cells (HUVEC) (Annual Report 1986), human epidermoid carcinoma cells (A-431), and human osteosarcoma cells (HOS) (Annual Report 1989). This work was extended to include EA hy.926 cells which are a hybrid of HUVEC and lung fibroblasts. Similar t-PA binding data were generated with HUVEC and EA hy.926 cells. Additional crosslinking studies between t-PA and the receptor were performed with 125I-labelled SASD (sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3- dithiopropionate), a photoreactive heterobifunctional reagent which in effect transfers the radioactive label from the ligand (t-PA) to the receptor on HUVEC. These studies confirmed previous indirect results which indicated that the receptor was in the molecular weight range of 54-65,000 as shown by SDS-PAGE. Isolation of the putative receptor was modified by passing cell lysates over a K1-K2 fragment conjugated affinity column in tandem with a t-PA affinity column. The K1-K2 fragment was obtained by limited trypsin digestion of t-PA and was shown to inhibit the binding of t-PA to HUEVC. t-PA was identified as a major binding protein by immunoblotting whereas scu-PA, PAI,1, and tubulin in addition to t-PA had been identified previously with the t-PA affinity column alone. Previous results had shown enhanced binding of t-PA to HUEVC in the presence of exogenously added scu-PA. Fragments of scu-PA were obtained from Abbott Labs and enhancement activity wash shown to be contained in the EGF region of scu-PA. Furthermore, antibodies to scu- PA and PAI-1 inhibited binding of t-PA to HUVEC by 50% respectively whereas anti-tubulin was ineffective. These results suggest that there is interaction of t-PA with scu-PA bound to its high affinity receptor on HUEVC as well as with PAI-1 in the substratum and that these phenomenon may account for the putative t-PA binding observed.